CELL BIOLOGY OF TRANSMEMBRANE SIGNALING PROTEINS

这项研究的长期目标是阐明小说 管理信息交换的机制 细胞。这里特别令人感兴趣的是 受体酪氨酸激酶的跨膜配体。这项建议 重点放在神经调节蛋白，一个多能细胞到细胞的家族 信号分子。神经调节蛋白及其受体，它们 受体酪氨酸激酶在发育过程中起着重要作用 神经系统和心脏内的决策，它们是 也与乳腺癌的发病机制有关。许多人 神经调节蛋白是以跨膜蛋白的形式合成的。尽管它 是跨膜中性调节蛋白的胞外结构域， 激活它们的同源酪氨酸激酶受体，胞浆 结构域序列更加保守(87%的同源性 人和鸡)。这种严格的序列守恒 强烈提示胞质结构域参与了 对神经调节蛋白的生物学行为至关重要的功能。 令人惊讶的是，中性调节蛋白胞浆结构域在很大程度上 在以往的研究中被忽视，因此其功能目前尚不清楚。 此外，人们对细胞质结构域知之甚少。 酪氨酸受体的其他跨膜配体的功能 激活剂。 与胞浆相互作用的两个非受体蛋白激酶 现在，酵母已经鉴定了神经调节蛋白的结构域-- 混合筛查。这些新的结果支持这样一种观点，即 神经调节蛋白胞质结构域在调控通路中的作用 是神经调节蛋白生物学的中心。身体和功能 神经调节蛋白与这些已识别的激酶的相互作用将是 在哺乳动物细胞中确定以确认这一假设的作用 对于中性调节蛋白胞质区域。免疫共沉淀， 培养细胞的免疫细胞化学和磷酸化分析 将用于这些第一批中性调节蛋白胞质结构域的研究 与激酶的相互作用。免疫组织化学将用于 确定神经调节蛋白和相互作用的激酶是否是共同的 在发育中的和成年的运动神经元和感觉神经元中表达。至 确定神经调节蛋白本身是否起到受体的作用， 实验将测试影响中性调素的事件是否 胞外区调控NeuRegin与细胞外信号通路的相互作用 鉴定出了激活酶。以确定更多的调节蛋白质 细胞质结构域功能，将进一步进行双杂交筛选 指挥。拟议的研究有望产生新的 对调节发育和发育的分子机制的见解 神经系统、心脏和乳房的病理过程。

The long-term goal of this research is to elucidate novel mechanisms that regulate the exchange of information between cells. Of specific interest here are the biological functions for transmembrane ligands of receptor tyrosine kinases. This proposal focuses on the neuregulins, a family of multipotent cell-to-cell signaling molecules. The neuregulins and their receptors, which are receptor tyrosinekinases, play essential roles in developmental decision-making within the nervous system and heart, and they are also implicated in the pathogenesis of breast cancer. Many of the neuregulins are synthesized as transmembrane proteins. Although it is the extracellular domain of transmembrane neuregulins that activates their cognate tyrosinekinase receptors, the cytoplasmic domain sequence is more highly conserved (87% identical between humans and chickens). This stringent conservation of sequence strongly suggests involvement of the cytoplasmic domain in functions critical to the biological actions of neuregulins. Surprisingly, the neuregulin cytoplasmic domain has been largely ignored in prior studies, so its function are currently unknown. Furthermore, little is known regarding cytoplasmic domain functions for other transmembrane ligands of receptor tyrosine kinases. Two non-receptor protein kinases that interact with the cytoplasmic domain of the neuregulins have now been identified by yeast two- hybrid screening. These new results support the idea that the neuregulin cytoplasmic domain functions in regulatory pathways central to neuregulin biology. The physical and functional interactions of the neuregulins with these identified kinases will be determined in mammalian cells to confirm this hypothesized role for the neuregulin cytoplasmic domain. Co-immunoprecipitation, immunocytochemistry, and phosphorylation assays in cultured cells will be used in these first studies of neuregulin cytoplasmic domain interactions with kinases. Immunohistochemistry will be used to determine whether neuregulins and the interacting kinases are co- expressed in developing and adult motor and sensory neurons. To determine if neuregulins themselves function as receptors, experiments will test whether events affecting the neuregulin extracellular domain regulate the interactions of neuregulin and the identified kinases. To identify additional proteins mediating cytoplasmic domain function, further two-hybrid screening will be conducted. The proposed studies are expected to produce new insights into molecular mechanisms regulating developmental and pathological processes in the nervous system, heart, and breast.